Development and Applications of Fourier Transform Mass Spectrometry
Introduction
Fourier transform mass spectrometry (FTMS), often referred to as Fourier transform ion cyclotron resonance MS (FT-ICR MS), is an important analytic technique. In FTMS, ions are confined to a particular path by specially designed magnetic fields. The oscillation frequency of an ion is defined by the mass to charge (m/z) ratio of the ion. Radio frequency pulses excite the ions to more energetic orbits and the simultaneous decay of the ions is observed as a free induction decay (FID) signal. A Fourier transform is used to obtain the individual ion frequencies from the FID and the m/z ratios are obtained from the frequencies.
FTMS requires the interaction of numerous technologies. Initially, the instrument was created by marrying existing technologies to produce the first generation FTMS systems. Existing ICR systems were combined with principles taken from nuclear magnetic resonance (NMR) spectroscopy. The mass accuracy and resolution have incrementally improved since the early days of FTMS as magnet and electronic technology and ion trap design have improved. Fragmentation techniques can also be applied to FTMS, allowing MS/MS to be performed with these instruments.
FTMS provides extremely high mass resolution (typically near 100,000 with high end systems reaching over 1,000,000) which is extremely useful for many applications. The ability to obtain the exact mass of analytes makes FTMS a very powerful technique. The large m/z window of modern systems makes analysis of large (> 50,000 Daltons) molecules possible. Additionally, since all ions are detected simultaneously, relatively crude sample can be analyzed without requiring purification. The high performance of FTMS means a wide variety of systems benefit from being studies with this technique.
Development
FTMS was essentially created by marrying some aspects of NMR spectroscopy with existing ICR mass spectrometers in the mid 1970’s [1]. The underlying physics of FTMS had been studied since the 1930’s with the development of the cyclotron [2]. The technology was adapted as a mass spectrometry technique in the 1950’s with the omegatron. Early measurements of the masses of protons and electrons were preformed with these devices. Ions were forced into a cyclic path by magnetic fields. A radiation pulse of a specific radio frequency was applied, exciting ions whose frequency match the radio frequency applied. Since the frequency of the ions is related to the m/z ratio, the radiation effectively acts as a selector for m/z ratio. When the ions are excited, the radius of their path increases. At this larger radius, a collection plate was located and detected the excited ions. To scan over various m/z values, early experimenters varied the radio frequency or, more commonly, the magnetic field strength, in a similar manner as magnetic sector mass spectrometers [3]. The instruments in this era were slow, but provided very advanced mass resolution for the time, allowing more precise measurements of masses to be preformed.
The next step on the path to FTMS was the development of ICR mass spectrometers in the mid 1960’s. Essentially, these spectrometers were very similar to the existing omegatrons with the exception of the detector. Instead of a collision plate detector being used, the perturbations of a radio frequency bridge caused by the excited ions were measured as the signal [4]. Again, the magnetic field was scanned to provide a full spectrum. The technique was frequently used to characterize reactions involving ions since it can be used to determine concentrations, populations, and collision frequencies from the mass spectrum. The excitation of one ion would have effects on others, giving insight into the energetic and kinetic properties of the reaction [5]. Early tandem MS experiments were also preformed using this technique, which was initially called double resonance spectroscopy [6]. One advantage in using an ICR mass spectrometer is the use of the ion trap allows experimenters to build up the number of ions present over time, before detection. If a sample had too low of a concentration to be seen in other forms of MS, ICR (and eventually, FTMS) could be used. Ions could be collected in the trap over a longer time scale, and then detected, effectively boosting the signal created by a given sample [1].
Similarly to NMR spectroscopy at this point in time, the technique was limited by single frequency monitoring. Obtaining full spectra was time consuming and tedious. The use of the Fourier transform revolutionized both techniques, although the application to mass spectrometry lagged behind that NMR applications for some time. The Fourier transform essentially takes a time dependant function composed of multiple oscillation frequencies and converts it into a frequency dependant function. Essentially, a signal produced by oscillations at many different frequencies is collected over time. After the Fourier transform, the signal is transformed into a function with peaks at the frequencies of the oscillators which produced the signal [7].
FT mass spectroscopy is an ICR spectrometer which uses broadband radio frequency radiation instead of a single frequency. The ions are all simultaneously excited, and their signal is also collected simultaneously. After the Fourier transform, their individual frequencies are obtained and converted to the m/z ratios. The application of the Fourier transform allows the entire spectrum to be collected during one excitation instead of requiring many single ion scans [5]. This allows more spectra to be collected, resulting in more sensitive measurement than earlier ICR mass spectrometers, greatly improving their utility.
Since the application of the Fourier transform, there have been two main approaches to improving the FTMS. One approach is the application of more powerful magnets. The oscillation frequency is proportional to the magnetic field strength, meaning that for two ions of constant m/z ratios, a stronger magnetic field will produce frequencies with a larger difference than a weaker magnet. Additionally, the upper mass limit for a given ion trap geometry is also proportional to the magnetic field strength. Stronger fields also allow higher kinetic energies to be reached, which is important for tandem mass spectrometry [8]. The other major avenue in instrumental advancement has been new ion traps. A better designed trap with superior geometry obtains better performance from a given magnetic field. A wide variety of ion traps and spectrometer geometries have been developed [9, 10]. The two approaches can be seen in two contrasting papers. Soon after the use of 9.4 Tesla magnets became feasible in the mid 1990’s, a group replaced a weaker magnet with a stronger one and greatly improved performance, obtaining a mass resolution of 50,000 [11]. In 2011, another group obtained over 800,000 mass resolution using the same strength magnet with a better designed instrument [12].
Tandem mass spectroscopy has been performed since the development of the first ICR mass spectrometers. Modern devices can often be fitted with multiple options for fragmentation. Collision induced decay (CID) was first used in an FTMS in 1978 [13]. In CID, precursor ions are accelerated through a cloud of gas. Each collision with a gas molecule imparts some energy to the ions, gradually building the internal energy of the ions until they fragment. Several different types of CID can be done, including multiple types of on resonance and off resonance CID [5]. A more recently developed fragmentation technique is electron capture dissociation (ECD). In ECD, low energy electrons are captured by ions, reducing the charge state by 1. When the electron is captured, bond cleavage can occur. The fragmentation pathways are quite different when compared to other fragmentation techniques, producing unique fragment ion spectra [14]. In addition to CID and ECD, there are several more fragmentation techniques available, including UV photodissociation, blackbody infrared radiative dissociation, and surface induced dissociation [5].
An interesting advantage obtained when doing tandem MS with an FTMS is the opportunity to perform repeated fragmentations. For several fragmentation techniques, the entire process can occur in the ion trap. Fragment ions simply shift their oscillation frequency after fragmentation. As a result, a fragment ion can be selected and refragmented, effectively performing MS/MS/MS. For extremely large molecules, this can be advantageous for structural and identification purposes [15]. The same fragmentation technique can be used multiple times, or several techniques can be applied in series depending on which fragmentation pathways are desirable for the experiment.
Virtually every widespread ionization technique has been coupled with an FTMS as some point including electron ionization (EI), chemical ionization (CI), several types of electrospray ionization (ESI), matrix assisted laser desorption ionization (MALDI), and atmospheric pressure laser ionization (APLI) [1]. Frequently, there is a huge advantage in using an ionization source that can produce multiply charged peaks, specifically ESI. When multiply charged ions are obtained, the effective mass range of the FTMS is greatly increased. High mass ions which would not be in a spectrometer’s mass range may be shifted to lower m/z ratios when they have a higher charge state [16]. For example, an ion with a mass of 50,000 would have m/z = 10,000 with a charge state of 5 and m/z = 5,000 with a charge state of 10. This expansion of the effective mass range of the spectrometer with multiply charged peaks can be of great use when working with high mass analytes.
The high versatility and performance of FT mass spectrometers can be seen in the commercial instruments available. In 2009, Bruker introduced the solariX FTMS, which can be fitted with a variety of magnet strengths ranging from 7 to 15 Tesla. Additionally, this model has an improved ion trap design which, when combined with the higher magnetic strengths, improves mass resolution to over 1,000,000. A variety of ion sources can be purchased, including MALDI, ETD, and several atmospheric pressure ionization techniques. The solariX is also capable of performing multiple fragmentation techniques such as CID and ECD [17]. This instrument is representative of the flexibility and resolving power possible with FT mass spectrometry.
Applications
The extremely high performance of modern FT mass spectrometers results in a wide variety of interesting and powerful applications. The relative ease with which these instruments can analyze fairly crude mixtures, operate over a wide mass range, and provide exact mass accuracy has allowed studies of previously difficult to characterize systems to be preformed [5].
The field of proteomics has been an important application of FTMS. The size of many proteins makes analysis by other mass spectrometry techniques difficult, especially if accurate mass identification is a goal. There are two basic approaches to using mass spectrometry in proteomics. The “bottom up” approach involves digesting a protein into smaller peptides and analyzing those fragments. The “top down” approach involves the direct analysis of intact proteins. Previously, due to mass resolution limits and maximum m/z limits, the bottom up approach was frequently the sole option for researchers. However, with the availability of FTMS, the top down approach is becoming used more frequently [18].
In the bottom up approach of proteomics, proteins are chemically broken down into smaller peptides by one or more of the many protein digests available. The peptide fragments can then be analyzed by a variety of mass spectrometry techniques. FT mass spectrometry is frequently selected due to the advantages of high sensitivity and exact mass measurements [18]. An extremely successful early proteomic experiment was preformed on a prokaryote. In this experiment, the genome of the species was mapped, and the predicted protein population was created. A simulation what peptide chains would be produced by the digests selected was preformed, producing a list of peptides expected to be found in the samples. In the mass range of 500 to 4000 Daltons, there were over 60,000 predicted peptides, which represented over 99% of the original protein population. Two analysis techniques were used to ensure accuracy. The first and more traditional technique was the use of LC/MS/MS to produce a potential value for the mass of the peptide. This procedure requires a long separation (over two hours) and the collection and later recombination of fractions. The second technique used an LC-FT-ICR mass spectrometer. This technique did not require tandem MS, which has several advantages. Then number of spectra collected is greatly reduced. Instead of need to do a fragmentation of many peaks as in the first method, in the second, the mass is obtained directly from the first (and only) mass spectrum. Additionally, fragmentation can effectively hide post translational modifications, which are often preserved in the second, single step analysis [19].
In this initial study, a typical FTMS spectrum would provide over 1,500 accurate mass tags for peptides. Additionally, when compared to the first method, only 70% of the tags found in the first method were confirmed by the second. With the use of FTMS, the study was able to generate accurate mass tags for over 60% of the proteins predicted to be present in the bacteria [19]. A later study improved the procedure to improve this value to over 80% [20].
Conversely, the top down approach to proteomics analyzes intact proteins instead of protein digest products. The mass identification of extremely high molecular weigh proteins introduces additional challenges which demand the high performance of FTMS. Even with conventionally high resolution, peaks separated by one Dalton become difficult to resolve at very high masses. The extremely high resolution provided by modern FTMS instruments allows isotopic peaks to be resolved and exact mass determinations to be made. Internal calibrants take on additional importance. More powerful and homogenous magnets and better circuitry are required as the high mass proteins push the limits of modern instruments [18].
Studying the intact protein has several advantages. Sample preparation is reduced, as digests are no longer needed. Also, the lack of digests allows post translational modification to be persevered, allowing for the identification of phosphorylation or disulfide bridges. If tandem mass spectrometry is used, ECD is frequently employed since it can preserve these post translational modifications [18]. One recent paper used a coupled LC/FT-ICR mass spectrometer to find exact masses to within 10 ppm for proteins up to 70,000 Dalton. ESI was used to produce ions of higher charge state for the high mass proteins, resulting in lower m/z ratios. ECD was used as a soft fragmentation technique to preserve as much of the structure and modifications of the protein as possible during fragmentation. Numerous difficulties in analyzing large proteins were addressed in this procedure. At high mass, the isotope distribution become very complex and narrows at higher charge states. For example, the spacing of a 13C and 12C peak with z=1 is one Dalton, while with z=10, the spacing is only 0.1 Dalton. Additionally, the isotope distribution can be greatly effected by metal adducts for some proteins. Modifications like glycosylation, phosphorylation and other oxidative modifications can introduce even more complexity into the distribution of peaks produced by one protein. Of important note is that this analysis was preformed with the FTMS in line with the nanoscale LC instrument [21]. As magnet technology continues to improve, the upper mass limit should continue to increase, allowing larger and larger proteins to be studied with a top down approach.
Metabolomics is another field in which FTMS can provide impressive results. Metabolomics is the study of low mass molecules present in a system, most of which are produced from enzymatic reactions [23]. Samples studied in metabolomics often have many thousand analytes present, making formula identification difficult. Even at low masses (<200 Dalton) there can be many empirical formulae which can fit for an exact mass. Previously, tandem mass spectroscopy or elemental analysis would be required to determine which of the possible formulae is correct. In a sample with many thousand molecules, this additional step in analysis is not desirable. Instead, there have been attempts to combine the excellent mass resolution of FTMS with ion abundance measurements to determine the formula of a given analyte. This would require not only excellent accuracy for the mass, but also excellent accuracy for the ion intensities. The isotope abundance information can be analyzes to determine which of the exact mass matches are compatible with the measured isotopic distribution. Attempts have been made to do this using selected ion monitoring (SIM) stitching with some success [23]. The number of possible formula was reduced by 60 to 90%, making the identification of the empirical formula much simpler. While this technique still has some significant error due to the extremely accurate ion intensity data required, it is still promising for future metabolomic research.
One experiment is a terrific example of the powerful combination of the resolving power, mass accuracy, and lack of sample preparation that FTMS provides. A sample of crude oil was diluted and spiked with a small amount of acetic acid. This was the entire sample preparation. The sample was analyzed with an FT-ICR mass spectrometer. Two hundred FIDs were collected, combined, and converted to a single mass spectrum. Over 11,000 resolved peaks were found in the resulting spectrum. The mass range for this experiment was approximately 300 to 900 Dalton [24]. The ability to resolve the extremely dense and numerous peaks in a single experiment from a fairly crude sample is an excellent illustration of the power FTMS can provide.
Conclusion
The beginnings of Fourier transform mass spectrometry can be traced back to the invention of the cyclotron in 1932 [2]. The omegatron and ICR mass spectrometer followed, but both were limited by single ion monitoring [3,4]. After the Fourier transform was applied to NMR spectroscopy, it was applied to ICR spectrometry. This allowed the simultaneous detection of all the ions present in the instrument, ushering in modern FT mass spectrometry [5].
Virtually every useful ionization and fragmentation method has been used at some point with an FTMS [5]. It compatible with many of the widely used techniques, but additional benefits are seen when ESI sources are used. ESI sources produce multiply charged ions, shifting the m/z ratio to a lower value than singly charged ions. This reduction in m/z can shift higher mass analytes into the m/z range of an FTMS [16]. Advances in FTMS design primarily arise from better designed ion traps and from more powerful magnets. Today, FT mass spectrometers have exceptionally high mass accuracy, mass resolution and sensitivity and often require little sample preparation or purification[5].
The high performance allows new and more powerful applications of mass spectrometry to be developed. Large molecule studies such as those done in proteomics and genomics benefit from these aspects of FTMS [18,20], as do studies of smaller molecules in metabolomics. As magnet technology becomes cheaper with time, FTMS should become more cost effective. Due to the high performance that can be obtained with FTMS [24], this technique should continue to grow in importance in many fields.
References
[1]. Comisarow, M. B., et al. Chem Phys. Lett. 1974, 25, 282-283. Link
[2]. Lawrence, E., et al. Phys. Rev. 1936, 50, 1131-1140. Link
[3]. Sommer, H., et al. Phys. Rev. 1951, 82, 697-702. Link
[4]. Wobscall, D. Rev. Sci. Instrum. 1965, 36, 466. Link
[5]. Marshall, A. G. Int. J. Mass Spectrom. 2000, 200, 331-356. Link
[6]. Anders, L. R. J. Chem. Phys. 1966, 45, 1062. Link
[7]. Fourier transform. http://en.wikipedia.org/wiki/Fourier_transform(accessed 4 December 2012). Part of Wikipedia. Link
[8]. Marshall, A. G., et al. Rapid Commun. Mass Spectrom. 1996, 10, 1819-1823. Link
[9]. Kaiser, N. K., et al. Anal. Chem. 2011, 83, 6907-6910. Link
[10]. Nikolaev, E. N., et al. J. Am. Soc. Mass Spectrom. 2011, 22, 1125-1133. Link
[11]. Senko, M. W., et al. Rapid Commun. Mass Spectrom. 1996, 10, 1824-1828.Link
[12]. Kaiser, N. K., et al. J. Am. Soc. Mass Spectrom. 2011, 22, 1343-1351. Link
[13]. Cody, R. B., et al. Anal. Chem. 1982, 54, 96-101. Link
[14]. Zubarev, R. A, et al. J. Am. Chem. Soc. 1998, 120, 3265-3266. Link
[15]. Huang, X., et al. Anal. Chim. Acta. 2008, 615, 124-135. Link
[16]. Henry, K. D., et al. Proc. Natl. Acad. Sci. U. S. A. 1989, 86, 9075-9078. Link
[17]. Bruker solariX Technical Details. http://www.bruker.com/products/mass-spectrometry-and-separations/ftms/solarix/technical-details.html (accessed 4 December 2012). Part of Bruker. Link
[18]. Bogdanoc, B., et al. Mass Spectrom. Rev. 2005, 24, 168-200. Link
[19]. Smith, R., D., et al. Proteomics. 2002, 2, 513-523.Link
[20]. Pasa-Tolic, L., et al. J. Mass Spectrom. 2002, 37, 1185-1198. Link
[21]. Timton, J. D., et al. Anal. Chem. 2012, 84, 2111-2117. Link
[22]. Marshall, A. G., et al. Mass Spectrom. Rev. 1998, 17, 1-35. Link
[23]. Weber, R. J. M., et al. Anal. Chem. 2011, 83, 3737-3743. Link
[24]. Hughey, C. A., et al. Anal. Chem. 2002, 74, 4145-4149. Link
Development and Applications of Fourier Transform Mass Spectrometry
Introduction
Fourier transform mass spectrometry (FTMS), often referred to as Fourier transform ion cyclotron resonance MS (FT-ICR MS), is an important analytic technique. In FTMS, ions are confined to a particular path by specially designed magnetic fields. The oscillation frequency of an ion is defined by the mass to charge (m/z) ratio of the ion. Radio frequency pulses excite the ions to more energetic orbits and the simultaneous decay of the ions is observed as a free induction decay (FID) signal. A Fourier transform is used to obtain the individual ion frequencies from the FID and the m/z ratios are obtained from the frequencies.
FTMS requires the interaction of numerous technologies. Initially, the instrument was created by marrying existing technologies to produce the first generation FTMS systems. Existing ICR systems were combined with principles taken from nuclear magnetic resonance (NMR) spectroscopy. The mass accuracy and resolution have incrementally improved since the early days of FTMS as magnet and electronic technology and ion trap design have improved. Fragmentation techniques can also be applied to FTMS, allowing MS/MS to be performed with these instruments.
FTMS provides extremely high mass resolution (typically near 100,000 with high end systems reaching over 1,000,000) which is extremely useful for many applications. The ability to obtain the exact mass of analytes makes FTMS a very powerful technique. The large m/z window of modern systems makes analysis of large (> 50,000 Daltons) molecules possible. Additionally, since all ions are detected simultaneously, relatively crude sample can be analyzed without requiring purification. The high performance of FTMS means a wide variety of systems benefit from being studies with this technique.
Development
FTMS was essentially created by marrying some aspects of NMR spectroscopy with existing ICR mass spectrometers in the mid 1970’s [1]. The underlying physics of FTMS had been studied since the 1930’s with the development of the cyclotron [2]. The technology was adapted as a mass spectrometry technique in the 1950’s with the omegatron. Early measurements of the masses of protons and electrons were preformed with these devices. Ions were forced into a cyclic path by magnetic fields. A radiation pulse of a specific radio frequency was applied, exciting ions whose frequency match the radio frequency applied. Since the frequency of the ions is related to the m/z ratio, the radiation effectively acts as a selector for m/z ratio. When the ions are excited, the radius of their path increases. At this larger radius, a collection plate was located and detected the excited ions. To scan over various m/z values, early experimenters varied the radio frequency or, more commonly, the magnetic field strength, in a similar manner as magnetic sector mass spectrometers [3]. The instruments in this era were slow, but provided very advanced mass resolution for the time, allowing more precise measurements of masses to be preformed.
The next step on the path to FTMS was the development of ICR mass spectrometers in the mid 1960’s. Essentially, these spectrometers were very similar to the existing omegatrons with the exception of the detector. Instead of a collision plate detector being used, the perturbations of a radio frequency bridge caused by the excited ions were measured as the signal [4]. Again, the magnetic field was scanned to provide a full spectrum. The technique was frequently used to characterize reactions involving ions since it can be used to determine concentrations, populations, and collision frequencies from the mass spectrum. The excitation of one ion would have effects on others, giving insight into the energetic and kinetic properties of the reaction [5]. Early tandem MS experiments were also preformed using this technique, which was initially called double resonance spectroscopy [6]. One advantage in using an ICR mass spectrometer is the use of the ion trap allows experimenters to build up the number of ions present over time, before detection. If a sample had too low of a concentration to be seen in other forms of MS, ICR (and eventually, FTMS) could be used. Ions could be collected in the trap over a longer time scale, and then detected, effectively boosting the signal created by a given sample [1].
Similarly to NMR spectroscopy at this point in time, the technique was limited by single frequency monitoring. Obtaining full spectra was time consuming and tedious. The use of the Fourier transform revolutionized both techniques, although the application to mass spectrometry lagged behind that NMR applications for some time. The Fourier transform essentially takes a time dependant function composed of multiple oscillation frequencies and converts it into a frequency dependant function. Essentially, a signal produced by oscillations at many different frequencies is collected over time. After the Fourier transform, the signal is transformed into a function with peaks at the frequencies of the oscillators which produced the signal [7].
FT mass spectroscopy is an ICR spectrometer which uses broadband radio frequency radiation instead of a single frequency. The ions are all simultaneously excited, and their signal is also collected simultaneously. After the Fourier transform, their individual frequencies are obtained and converted to the m/z ratios. The application of the Fourier transform allows the entire spectrum to be collected during one excitation instead of requiring many single ion scans [5]. This allows more spectra to be collected, resulting in more sensitive measurement than earlier ICR mass spectrometers, greatly improving their utility.
Since the application of the Fourier transform, there have been two main approaches to improving the FTMS. One approach is the application of more powerful magnets. The oscillation frequency is proportional to the magnetic field strength, meaning that for two ions of constant m/z ratios, a stronger magnetic field will produce frequencies with a larger difference than a weaker magnet. Additionally, the upper mass limit for a given ion trap geometry is also proportional to the magnetic field strength. Stronger fields also allow higher kinetic energies to be reached, which is important for tandem mass spectrometry [8]. The other major avenue in instrumental advancement has been new ion traps. A better designed trap with superior geometry obtains better performance from a given magnetic field. A wide variety of ion traps and spectrometer geometries have been developed [9, 10]. The two approaches can be seen in two contrasting papers. Soon after the use of 9.4 Tesla magnets became feasible in the mid 1990’s, a group replaced a weaker magnet with a stronger one and greatly improved performance, obtaining a mass resolution of 50,000 [11]. In 2011, another group obtained over 800,000 mass resolution using the same strength magnet with a better designed instrument [12].
Tandem mass spectroscopy has been performed since the development of the first ICR mass spectrometers. Modern devices can often be fitted with multiple options for fragmentation. Collision induced decay (CID) was first used in an FTMS in 1978 [13]. In CID, precursor ions are accelerated through a cloud of gas. Each collision with a gas molecule imparts some energy to the ions, gradually building the internal energy of the ions until they fragment. Several different types of CID can be done, including multiple types of on resonance and off resonance CID [5]. A more recently developed fragmentation technique is electron capture dissociation (ECD). In ECD, low energy electrons are captured by ions, reducing the charge state by 1. When the electron is captured, bond cleavage can occur. The fragmentation pathways are quite different when compared to other fragmentation techniques, producing unique fragment ion spectra [14]. In addition to CID and ECD, there are several more fragmentation techniques available, including UV photodissociation, blackbody infrared radiative dissociation, and surface induced dissociation [5].
An interesting advantage obtained when doing tandem MS with an FTMS is the opportunity to perform repeated fragmentations. For several fragmentation techniques, the entire process can occur in the ion trap. Fragment ions simply shift their oscillation frequency after fragmentation. As a result, a fragment ion can be selected and refragmented, effectively performing MS/MS/MS. For extremely large molecules, this can be advantageous for structural and identification purposes [15]. The same fragmentation technique can be used multiple times, or several techniques can be applied in series depending on which fragmentation pathways are desirable for the experiment.
Virtually every widespread ionization technique has been coupled with an FTMS as some point including electron ionization (EI), chemical ionization (CI), several types of electrospray ionization (ESI), matrix assisted laser desorption ionization (MALDI), and atmospheric pressure laser ionization (APLI) [1]. Frequently, there is a huge advantage in using an ionization source that can produce multiply charged peaks, specifically ESI. When multiply charged ions are obtained, the effective mass range of the FTMS is greatly increased. High mass ions which would not be in a spectrometer’s mass range may be shifted to lower m/z ratios when they have a higher charge state [16]. For example, an ion with a mass of 50,000 would have m/z = 10,000 with a charge state of 5 and m/z = 5,000 with a charge state of 10. This expansion of the effective mass range of the spectrometer with multiply charged peaks can be of great use when working with high mass analytes.
The high versatility and performance of FT mass spectrometers can be seen in the commercial instruments available. In 2009, Bruker introduced the solariX FTMS, which can be fitted with a variety of magnet strengths ranging from 7 to 15 Tesla. Additionally, this model has an improved ion trap design which, when combined with the higher magnetic strengths, improves mass resolution to over 1,000,000. A variety of ion sources can be purchased, including MALDI, ETD, and several atmospheric pressure ionization techniques. The solariX is also capable of performing multiple fragmentation techniques such as CID and ECD [17]. This instrument is representative of the flexibility and resolving power possible with FT mass spectrometry.
Applications
The extremely high performance of modern FT mass spectrometers results in a wide variety of interesting and powerful applications. The relative ease with which these instruments can analyze fairly crude mixtures, operate over a wide mass range, and provide exact mass accuracy has allowed studies of previously difficult to characterize systems to be preformed [5].
The field of proteomics has been an important application of FTMS. The size of many proteins makes analysis by other mass spectrometry techniques difficult, especially if accurate mass identification is a goal. There are two basic approaches to using mass spectrometry in proteomics. The “bottom up” approach involves digesting a protein into smaller peptides and analyzing those fragments. The “top down” approach involves the direct analysis of intact proteins. Previously, due to mass resolution limits and maximum m/z limits, the bottom up approach was frequently the sole option for researchers. However, with the availability of FTMS, the top down approach is becoming used more frequently [18].
In the bottom up approach of proteomics, proteins are chemically broken down into smaller peptides by one or more of the many protein digests available. The peptide fragments can then be analyzed by a variety of mass spectrometry techniques. FT mass spectrometry is frequently selected due to the advantages of high sensitivity and exact mass measurements [18]. An extremely successful early proteomic experiment was preformed on a prokaryote. In this experiment, the genome of the species was mapped, and the predicted protein population was created. A simulation what peptide chains would be produced by the digests selected was preformed, producing a list of peptides expected to be found in the samples. In the mass range of 500 to 4000 Daltons, there were over 60,000 predicted peptides, which represented over 99% of the original protein population. Two analysis techniques were used to ensure accuracy. The first and more traditional technique was the use of LC/MS/MS to produce a potential value for the mass of the peptide. This procedure requires a long separation (over two hours) and the collection and later recombination of fractions. The second technique used an LC-FT-ICR mass spectrometer. This technique did not require tandem MS, which has several advantages. Then number of spectra collected is greatly reduced. Instead of need to do a fragmentation of many peaks as in the first method, in the second, the mass is obtained directly from the first (and only) mass spectrum. Additionally, fragmentation can effectively hide post translational modifications, which are often preserved in the second, single step analysis [19].
In this initial study, a typical FTMS spectrum would provide over 1,500 accurate mass tags for peptides. Additionally, when compared to the first method, only 70% of the tags found in the first method were confirmed by the second. With the use of FTMS, the study was able to generate accurate mass tags for over 60% of the proteins predicted to be present in the bacteria [19]. A later study improved the procedure to improve this value to over 80% [20].
Conversely, the top down approach to proteomics analyzes intact proteins instead of protein digest products. The mass identification of extremely high molecular weigh proteins introduces additional challenges which demand the high performance of FTMS. Even with conventionally high resolution, peaks separated by one Dalton become difficult to resolve at very high masses. The extremely high resolution provided by modern FTMS instruments allows isotopic peaks to be resolved and exact mass determinations to be made. Internal calibrants take on additional importance. More powerful and homogenous magnets and better circuitry are required as the high mass proteins push the limits of modern instruments [18].
Studying the intact protein has several advantages. Sample preparation is reduced, as digests are no longer needed. Also, the lack of digests allows post translational modification to be persevered, allowing for the identification of phosphorylation or disulfide bridges. If tandem mass spectrometry is used, ECD is frequently employed since it can preserve these post translational modifications [18]. One recent paper used a coupled LC/FT-ICR mass spectrometer to find exact masses to within 10 ppm for proteins up to 70,000 Dalton. ESI was used to produce ions of higher charge state for the high mass proteins, resulting in lower m/z ratios. ECD was used as a soft fragmentation technique to preserve as much of the structure and modifications of the protein as possible during fragmentation. Numerous difficulties in analyzing large proteins were addressed in this procedure. At high mass, the isotope distribution become very complex and narrows at higher charge states. For example, the spacing of a 13C and 12C peak with z=1 is one Dalton, while with z=10, the spacing is only 0.1 Dalton. Additionally, the isotope distribution can be greatly effected by metal adducts for some proteins. Modifications like glycosylation, phosphorylation and other oxidative modifications can introduce even more complexity into the distribution of peaks produced by one protein. Of important note is that this analysis was preformed with the FTMS in line with the nanoscale LC instrument [21]. As magnet technology continues to improve, the upper mass limit should continue to increase, allowing larger and larger proteins to be studied with a top down approach.
Metabolomics is another field in which FTMS can provide impressive results. Metabolomics is the study of low mass molecules present in a system, most of which are produced from enzymatic reactions [23]. Samples studied in metabolomics often have many thousand analytes present, making formula identification difficult. Even at low masses (<200 Dalton) there can be many empirical formulae which can fit for an exact mass. Previously, tandem mass spectroscopy or elemental analysis would be required to determine which of the possible formulae is correct. In a sample with many thousand molecules, this additional step in analysis is not desirable. Instead, there have been attempts to combine the excellent mass resolution of FTMS with ion abundance measurements to determine the formula of a given analyte. This would require not only excellent accuracy for the mass, but also excellent accuracy for the ion intensities. The isotope abundance information can be analyzes to determine which of the exact mass matches are compatible with the measured isotopic distribution. Attempts have been made to do this using selected ion monitoring (SIM) stitching with some success [23]. The number of possible formula was reduced by 60 to 90%, making the identification of the empirical formula much simpler. While this technique still has some significant error due to the extremely accurate ion intensity data required, it is still promising for future metabolomic research.
One experiment is a terrific example of the powerful combination of the resolving power, mass accuracy, and lack of sample preparation that FTMS provides. A sample of crude oil was diluted and spiked with a small amount of acetic acid. This was the entire sample preparation. The sample was analyzed with an FT-ICR mass spectrometer. Two hundred FIDs were collected, combined, and converted to a single mass spectrum. Over 11,000 resolved peaks were found in the resulting spectrum. The mass range for this experiment was approximately 300 to 900 Dalton [24]. The ability to resolve the extremely dense and numerous peaks in a single experiment from a fairly crude sample is an excellent illustration of the power FTMS can provide.
Conclusion
The beginnings of Fourier transform mass spectrometry can be traced back to the invention of the cyclotron in 1932 [2]. The omegatron and ICR mass spectrometer followed, but both were limited by single ion monitoring [3,4]. After the Fourier transform was applied to NMR spectroscopy, it was applied to ICR spectrometry. This allowed the simultaneous detection of all the ions present in the instrument, ushering in modern FT mass spectrometry [5].
Virtually every useful ionization and fragmentation method has been used at some point with an FTMS [5]. It compatible with many of the widely used techniques, but additional benefits are seen when ESI sources are used. ESI sources produce multiply charged ions, shifting the m/z ratio to a lower value than singly charged ions. This reduction in m/z can shift higher mass analytes into the m/z range of an FTMS [16]. Advances in FTMS design primarily arise from better designed ion traps and from more powerful magnets. Today, FT mass spectrometers have exceptionally high mass accuracy, mass resolution and sensitivity and often require little sample preparation or purification[5].
The high performance allows new and more powerful applications of mass spectrometry to be developed. Large molecule studies such as those done in proteomics and genomics benefit from these aspects of FTMS [18,20], as do studies of smaller molecules in metabolomics. As magnet technology becomes cheaper with time, FTMS should become more cost effective. Due to the high performance that can be obtained with FTMS [24], this technique should continue to grow in importance in many fields.
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